Single-Molecule Sequencing of the C9orf72 Repeat Expansion in Patient iPSCs

A hexanucleotide GGGGCC repeat expansion in the C9orf72 gene is the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). C9orf72 repeat expansions are currently identified with long-range PCR or Southern blot for clinical and research purposes, but these methods lack accuracy and sensitivity. The GC-rich and repetitive content of the region cannot be amplified by PCR, which leads traditional sequencing approaches to fail. We turned instead to PacBio single-molecule sequencing to detect and size the C9orf72 repeat expansion without amplification. We isolated high molecular weight genomic DNA from patient-derived iPSCs of varying repeat lengths and then excised the region containing the C9orf72 repeat expansion from naked DNA with a CRISPR/Cas9 system. We added adapters to the cut ends, capturing the target region for sequencing on PacBio’s Sequel, Sequel II, or Sequel IIe. This approach enriches the C9orf72 repeat region without amplification and allows the repeat expansion to be consistently and accurately sized, even for repeats in the thousands. Key features • This protocol is adapted from PacBio’s previous “no-amp targeted sequencing utilizing the CRISPR-Cas9 system.” • Optimized for sizing C9orf72 repeat expansions in patient-derived iPSCs and applicable to DNA from any cell type, blood, or tissue. • Requires high molecular weight naked DNA. • Compatible with Sequel I and II but not Revio.

This protocol is used in: Sci Rep (2024), DOI: 10.1038/s41598-023-50667-3 A hexanucleotide GGGGCC repeat expansion in the C9orf72 gene is the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD).C9orf72 repeat expansions are currently identified with long-range PCR or Southern blot for clinical and research purposes, but these methods lack accuracy and sensitivity.The GC-rich and repetitive content of the region cannot be amplified by PCR, which leads traditional sequencing approaches to fail.We turned instead to PacBio single-molecule sequencing to detect and size the C9orf72 repeat expansion without amplification.We isolated high molecular weight genomic DNA from patient-derived iPSCs of varying repeat lengths and then excised the region containing the C9orf72 repeat expansion from naked DNA with a CRISPR/Cas9 system.We added adapters to the cut ends, capturing the target region for sequencing on PacBio's Sequel, Sequel II, or Sequel IIe.This approach enriches the C9orf72 repeat region without amplification and allows the repeat expansion to be consistently and accurately sized, even for repeats in the thousands.Note: We skip this step.Instead, we evaluate DNA quality with a 1% agarose gel as described in steps B4-7 (Figure 1). 3. Prepare a 1% agarose gel with SYBR TM Safe DNA gel stain [17].4. Add 2 μL of genomic DNA, 7 μL of water, and 1 μL of 6× gel loading dye to a PCR tube. 5. Pipette-mix gently and load the diluted DNA into the 1% gel.6. Run the gel at 100 V for 1 h.7. Image the gel and check for any smearing in your samples.Samples with a single band above 10 kb should be included.Samples that have smearing down the gel, even if they have the >10 kb band, indicate DNA shearing and should be excluded from the protocol.In the case of shearing, fresh genomic DNA samples should be collected.

C. Prepare reagents
Prior to starting the library preparation, prepare the PacBio barcoded adapters for multiplexing and the carrier DNA. 1.To prepare the PacBio barcoded adapters for multiplexing, resuspend the barcoded adapter oligos in nuclease-free water to 100 μM. 2. Add the following reagents, in order, to a PCR tube to make 20 μM stocks of barcoded adapters: 100 μM barcoded adapter oligo 10 μL 10× annealing buffer 5 μL Nuclease-free water 35 μL 3. Place the 20 μM stocks in a thermal cycler and run the following protocol: 95 °C for 5 min Decrease to 25 °C, ramping down at the maximum cooling rate 4 °C hold 4. Store stocks at -20 °C. 5. To prepare the carrier DNA (1 kb DNA ladder), add 5 μL of ladder to 45 μL of elution buffer in a PCR tube to dilute the carrier DNA to a concentration of 50 ng/μL.6. Store at -20 °C.

D. Dephosphorylate the genomic DNA
1.In a PCR tube, dilute each genomic DNA sample to 5 μg with nuclease-free water for a total volume of 68 μL.
Note: When multiplexing five samples, 5 μg per sample is the optimal input.If using a different number of samples (1)(2)(3)(4)(5)(6)(7)(8)(9)(10) for this protocol, the minimum total DNA input is 5 μg and the maximum total DNA input is 25 μg.Each multiplexed sample should have equimolar input amounts.F. Prepare the gRNA/Cas9 complex 1. Add the following reagents, in order, to a LoBind microcentrifuge tube to make a master mix.Add 1 volume of each reagent per sample with 10% overage: Nuclease-free water 7 μL NEBuffer 3.1 2 μL Cas9 nuclease 2 μL Diluted sgRNAs (from section E) 8 μL 2. Pipette-mix the reaction.3. Briefly spin down the tube in a mini centrifuge.4. Transfer the master mix to a PCR tube and place it in a thermal cycler.5. Incubate at 37 °C for 10 min.6. Place the gRNA/Cas9 complex on ice.

Figure 1 .
Figure 1.1% Agarose gel evaluation of genomic DNA quality.As a quick quality check, we run isolated DNA on a 1% agarose gel prior to initiating CRISPR isolation of the target region.We include only DNA that has high molecular weight (i.e., has no smearing).(A, B) Examples of genomic DNA samples extracted from C9orf72 patient iPSC lines and run on a 1% agarose gel to evaluate sample quality.(A) Samples 1, 2, 4, and 5 (A) and 6 (B) show no smearing and would be acceptable for use in the protocol.Samples 3 (A) and 7 (B) show significant smearing and therefore we recommend being excluded from this protocol.This is a quick quality check.Sample quality can be confirmed with higher accuracy by the Agilent FEMTO Pulse System, the Bio-Rad CHEF Mapper XA Pulsed Field Electrophoresis System, or the Sage Science Pippin Pulse Electrophoresis Power Supply.

2 .
Add the following reagents, in order, to a LoBind microcentrifuge tube to make a master mix.Add 1 volume of each reagent per sample with 10% overage: NEBuffer 8 μL rSAP 4 μL 3. Add 12 μL of dephosphorylation master mix to each sample for a total volume of 80 μL. 4. Invert the tubes 20 times to mix.Note: Do not vortex or flick the tube to avoid shearing the DNA. 5. Briefly spin down the tubes in a mini centrifuge.6. Place in the thermal cycler and run the following protocol: 37 °C for 1 h 65 °C for 10 min 4 °C hold E. Prepare the single-guide RNAs (sgRNAs)

Cite as: Tsai, Y.C. et al. (2024). Single-Molecule Sequencing of the C9orf72 Repeat Expansion in Patient iPSCs. Bio- protocol 14(17): e5060. DOI: 10.21769/BioProtoc.5060. 4 Published: Sep 05, 2024
1. Measure the concentration of your genomic DNA using the Qubit TM 1× dsDNA HS Assay kit and associated protocol[16].Note: Prior to measuring the concentrations, dilute genomic DNA 1:10.2. It is recommended to evaluate sample quality with the Agilent FEMTO Pulse System, the Bio-Rad CHEF Mapper XA Pulsed Field Electrophoresis System, or the Sage Science Pippin Pulse Electrophoresis Power Supply.Genomic DNA samples should have fragment sizes ≥ 50 kb.Significant on-target reads can still be achieved with fragment sizes <50 kb by increasing the amount of input DNA, but better results may be achieved by collecting a fresh sample to achieve fragment sizes ≥ 50 kb.